Active Anti-Inflammatory and Hypolipidemic Derivatives Of

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Article Active Anti-Inﬂammatory and Hypolipidemic Derivatives of Lorazepam

Panagiotis Theodosis-Nobelos 1,2, Georgios Papagiouvannis 1, Panos N. Kourounakis 1 and Eleni A. Rekka 1,*

1 Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki, 54124 Thessaloniki, Greece 2 Department of Pharmacy, School of Health Sciences, Frederick University, Nicosia 1036, Cyprus * Correspondence: [email protected]; Tel.: +30-231-099-7614

Academic Editor: Athina Geronikaki Received: 22 July 2019; Accepted: 2 September 2019; Published: 9 September 2019

Abstract: Novel derivatives of some non steroidal anti-inflammatory drugs, as well as of the antioxidants α-lipoic acid, trolox and (E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylic acid with lorazepam were synthesised by a straightforward method at satisfactory to high yields (40%–93%). All the tested derivatives strongly decreased lipidemic indices in rat plasma after Triton induced hyperlipidaemia. They also reduced acute inflammation and a number of them demonstrated lipoxygenase inhibitory activity. Those compounds acquiring antioxidant moiety were inhibitors of lipid peroxidation and radical scavengers. Therefore, the synthesised compounds may add to the current knowledge about multifunctional agents acting against various disorders implicating inflammation, dyslipidaemia and oxidative stress.

Keywords: non steroidal anti-inﬂammatory drugs; lorazepam derivatives; antioxidants; inﬂammation; hyperlipidemia; lipoxygenase inhibition

1. Introduction Lorazepam (7-chloro-5-(2-chlorophenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin-2-one) is a benzodiazepine derivative, used as anxiolytic, sedative, in status epilepticus and in the treatment of alcohol withdrawal. It is known that benzodiazepines act by potentiating the interaction of GABA with GABAA receptors. Biologic stress has been described as the non-specific adaptive response of the organism to various stimuli, physical, psychological or emotional, such as fear and anxiety [1]. We [1] and others [2] have shown that biologic stress induces oxidative stress, and that GABAergic modulation may offer protection against immobilization-induced stress and oxidative damage [2]. Moreover, it has been reported that the benzodiazepine midazolam protects against neuronal degeneration and apoptosis induced by biological and oxidative stress [3]. Alimentary dyslipidemia disturbed anxiety level and cognitive processes in mice [4] and a diet rich in saturated fat and fructose caused high serum cholesterol and triglyceride concentrations and induced a condition in rats similar to the human metabolic syndrome. This condition was accompanied by anxiety-related behaviour, which correlated significantly with oxidative stress. In addition, the degree of lipid peroxidation correlated well with the metabolic effects of the diet [5]. Finally, a number of benzodiazepine derivatives reduced hyperalgesia in rats in a dose-dependent manner, using the carrageenan-induced method [6]. Taking the above evidence into consideration, in this investigation we report the synthesis and biological evaluation of esters of lorazepam with five classical NSAIDs and the antioxidants α-lipoic acid ((R)-5-(1,2-dithiolan-3-yl)pentanoic acid), trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and (E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylic acid (BHA). The aim of this work was to

Molecules 2019, 24, 3277; doi:10.3390/molecules24183277 www.mdpi.com/journal/molecules Molecules 2019, 24, 3277 2 of 12 investigate whether such derivatives would combine hypolipidemic and anti-inflammatory activity and to examine their potential lipoxygenase inhibitory, antioxidant and radical scavenging activities. We have demonstrated that a number of lorazepam derivatives possess hypolipidemic action [7]. Furthermore, alpha-lipoic acid reduced inflammation and oxidative stress in patients with metabolic syndrome [8]. We have also reported that trolox and cinnamic acid derivatives demonstrated antioxidant, anti-inflammatory and hypolipidemic activities [9]. Lastly, indole-3-acetic acid, part of the indomethacin structure, was also used.

2. Results and Discussion

2.1. Synthesis Compounds 1–6, 8, 9 were synthesised by direct esteriﬁcation of the carboxylic group of the respectiveMolecules acids 2019 with, 24, x lorazepam, using N,N0-dicyclohexyl-carbodiimide (DCC), at room temperature 3 of 12 and high yields. For compound 7, carbonyldiimidazole (CDI) was used (Figure1).

H H O N O O N Cl O Cl HO R O + N A) DCC/DMAP/CH2Cl2/r.t. N R OH B) CDI/THF/r.t. Cl Cl

Yield No. RCOOH (%) O 1 Ibuprofen OH 74

O O 2 Naproxen 93 OH

O 3 Ketoprofen OH 85 O O OH 4 Indomethacin O 81 N O Cl O OH 5 Tolfenamic acid NH Cl 41

O 6 α-Lipoic acid 71 S OH S HO O 7 Trolox 85 O OH

O OH 8 BHA 40 HO

9 Indole-3-acetic acid O 40 N H

FigureFigure 1. Synthesis 1. Synthesis and and structures structures of the the examined examined compounds. compounds. r.t. r.t.

2.2. Biological Activity

2.2.1. In Vivo Experiments

Effect of Compounds on Acute Inflammation in Rats The effect of the synthesised compounds on acute inflammation, applying the carrageenan rat paw oedema model, as well as the anti-inflammatory activity of the parent NSAIDs, are shown in Table 1.

Molecules 2019, 24, 3277 3 of 12

2.2. Biological Activity

2.2.1. In Vivo Experiments

Effect of Compounds on Acute Inflammation in Rats The effect of the synthesised compounds on acute inflammation, applying the carrageenan rat paw oedema model, as well as the anti-inflammatory activity of the parent NSAIDs, are shown in Table1.

Table 1. Eﬀect of compounds 1–9, ibuprofen, naproxen, ketoprofen, indomethacin and tolfenamic acid on carrageenan-induced rat paw oedema a.

Compound % Oedema Reduction 1 68 ** Ibuprofen 36 * 2 46 ** Naproxen 11 * 3 65 ** Ketoprofen 47 * 4 67 ** Indomethacin 42 ** 5 55 ** Tolfenamic acid 24 ** 6 39 * 7 56 * 8 37 * 9 43 ** a The effect on oedema is expressed as percent inhibition of oedema in comparison to controls, 3.5 h post administration. All compounds were administered intra peritonealy (i.p.) at 0.15 mmol/kg body weight. Significant difference from control: * p < 0.01, ** p < 0.001 (Student’s t test).

The carrageenan-induced paw oedema is a commonly and widely used model of acute inflammation. The early phase of carrageenan inflammation is characterised mainly by the release of histamine, serotonin and bradykinin. In the late phase, more than two hours after administration, the additional effects of neutrophil infiltration, prostaglandin production and pro-inflammatory cytokine release develop [10]. In this investigation, oedema was estimated 3.5 h after carrageenan administration. All compounds demonstrated increased anti-inflammatory activity. NSAID derivatives 1–5 were more potent than their individual parent acids. Especially, compound 2 was four times more active than naproxen. The activity of 1 and 5 were about two fold higher than ibuprofen and tolfenamic acid, respectively. Overall, these results indicate a further enhancement of the anti-inflammatory activity by the performed molecular modification. It has been found that diazepam reduces the number of inflammatory cells in the central nervous system [11], treatment with high diazepam doses decreases paw oedema after carrageenan-induced injury [12] and that benzodiazepine derivatives with imide substitution at the C3 of the benzodiazepine ring reduced up to 80% carrageenan rat paw hyperalgesia, in a dose dependent manner, and this activity was at least partly attributed to bradykinin B1 receptor antagonism [6]. Compounds 7 and 8 showed considerable anti-inflammatory activity. There is not any reported anti-inflammatory activity for trolox. Additionally, butylated hydroxytoluene (BHT), a well known antioxidant structurally similar to the parent acid BHA, lacks such action either in vivo [13], or in vitro [14]. Thus, the antioxidant activity alone does not seem to be entirely responsible for the anti-inflammatory activity. Furthermore, compounds 6 and 9 also inhibited paw oedema, although they do not express any antioxidant activity (part 2.2.2.1.). Indole-3-acetic acid has no reported anti-inflammatory action, while lipoic acid is only a weak anti-inflammatory agent at similar doses [15]. Again, it seems convincing that the lorazepam moiety contributes to increased anti-inflammatory activity. Molecules 2019, 24, 3277 4 of 12

Eﬀect of Compounds on Hyperlipidemia in Rats The eﬀect of the compounds under investigation on plasma total cholesterol, triglyceride and LDL-cholesterol levels, 24 h post injection, determined in rats after Triton-induced hyperlipidaemia is shown in Table2. Simvastatin, ibuprofen and naproxen are included for comparison.

Table 2. Eﬀect of the tested compounds, simvastatin, ibuprofen and naproxen on Triton WR1339 (tyloxapol) induced hyperlipidemia.

Dose i.p. % Reduction Compound (µmol/kg) TC a TG b LDL-C c 1 150 82 *** 65 *** 56 * 1 50 44 *** 57 ** 43 ** 2 50 60 *** 71 *** 42 *** 4 50 59 *** 59 *** 60 *** 5 50 56 *** 57 *** 48 *** 6 150 82 *** 41 ** 60 * 6 50 48 *** 39 *** 47 *** 7 150 72 *** 64 * 69 * 8 150 81 *** 66 *** 63 *** 8 50 59 *** 52 *** 44 ** Simvastatin 150 73 *** - 70 *** Ibuprofen 300 41 *** 38 *** 42 *** Naproxen 500 53 *** 44 *** 26 *** a TC: Total cholesterol; b TG: Triglycerides; c LDL-C: LDL cholesterol. Tyloxapol: 200 mg/kg, i.p. Signiﬁcant diﬀerence from hyperlipidemic control: * p < 0.01, ** p < 0.005, *** p < 0.001 (Student’s t test).

Tyloxapol (Triton WR1339) is a nonionic surfactant used to induce hyperlipidemia in experimental animals if administered parenterally. It leads to accumulation of triglycerides, VLDL- and LDL-cholesterol in plasma, reaching maximal effect 24 h after administration. These actions are due to inhibition of lipoprotein lipase and to stimulation of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase [9,16]. Compounds 1 and 6–8 were administered at 0.15mmol/kg and reduced greatly all lipidemic indices, e.g., total cholesterol reduction was at the range of 80%, comparable to that of simvastatin. For most compounds a lower dose, 0.05mmol/kg, was tested and found that still a very significant reduction of lipidemia was achieved, further indicating a dose dependent action. Compounds 1 and 2 were more active than ibuprofen and naproxen at 6–10 times lower dose. These results may be related to their high anti-inflammatory activity and partly may be related to a potential antioxidant effect.

2.2.2. In Vitro Experiments

Antioxidant Activity Considering that free radicals are implicated in inﬂammatory processes, the synthesised compounds were tested for antioxidant activity, expressed as inhibition of rat microsomal membrane lipid peroxidation induced by ferrous ascorbate, as well as interaction with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). The percent interaction with DPPH and the IC50 values of the active ﬁnal compounds and trolox from rat hepatic microsomal membrane lipid peroxidation, after 45 min of incubation are shown in Table3. Molecules 2019, 24, 3277 5 of 12

MoleculesTable 3.2019Interaction, 24, x of compounds 7, 8 and trolox, at various concentrations, with DPPH (200 µ 6 M)of 12a and their eﬀect on lipid peroxidation b. Table 3. Interaction of compounds 7, 8 and trolox, at various concentrations, with DPPH (200 µΜ) a and their effect on lipid peroxidationPercent Interaction b. with DPPH Inhibition of Lipid Compound Peroxidation IC50 (µM) Percent200 µ MInteraction 100withµM DPPH 50 µInhibitionM of Lipid Peroxidation Compound 7 200 µΜ91 100 µΜ 8250 µΜ 46IC50 (µΜ) 2.5 7 8 91 9082 4946 30 2.5 > 100 Trolox8 90 92 49 90 30 38 > 100 25 a AfterTrolox 30 min of incubation. 92 b After 45 min 90 of incubation. Trolox: 38 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic 25 acid.a All After determinations 30 min of were incubation. performed atb least After in triplicate 45 min and of standard incubation. deviation Trolox: is always 6-hydroxy-2,5,7,8- within 10% of the mean value. ± tetramethylchroman-2-carboxylic acid. All determinations were performed at least in triplicate and standard deviation is always within ± 10% of the mean value. The time course of lipid peroxidation inhibition, as aﬀected by various concentrations of 7 is The time course of lipid peroxidation inhibition, as affected by various concentrations of 7 is shownshown in Figurein Figure2. 2.

1.2 Compound 7 1.0 IC50 = 2.5 μΜ

0.8 Control 1 μΜ 0.6 2 μΜ 3 μΜ Α (535/600 nm) 0.4

0.2

0.0 0 5 10 15 20 25 30 35 40 45 Time (min)

Figure 2. Inhibition of lipid peroxidation, as affected by various concentrations of 7, in relation to time. Figure 2. Inhibition of lipid peroxidation, as affected by various concentrations of 7, in relation to Mosttime. compounds, except for 7 and 8, were practically inactive in these experiments. In the lipid peroxidationMost test,compounds, compound except8 showed for 7 and moderate 8, were practically activity. inactive This may in bethese due experiments. to the high In lipophilicity the lipid of compoundperoxidation8 (logP test,= compound7.4) which 8 reduced showed its moderate solubility activity. in the This aqueous may be reaction due toenvironment. the high lipophilicity However, it interactedof compound strongly 8 with (logP DPPH, = 7.4) in which a way reduced comparable its solubility to that of in trolox. the aqueous Compound reaction7 was environment. almost tenfold moreHowever, potent inhibitor it interacted of lipidstrongly peroxidation with DPPH, than in a trolox, way comparable a reference to antioxidant, that of trolox. and Compound an effective 7 was radical scavenger.almost tenfold It is possible more potent that the inhibitor hypolipidemic of lipid peroxidation and anti-inflammatory than trolox, a e referenceffects of theseantioxidant, compounds and are related,an effective at least radical partly, scavenger. to their antioxidant It is possible activity. that the The hypolipidemic lipoic acid derivative and anti-inflammatory6 was found effects inactive, of and thisthese is in accordancecompounds withare related, the observation at least partly, that to lipoic their acid antioxidant can scavenge activity. only The very lipoic reactive acid derivative radicals, 6 while the reducedwas found form, inactive, 6,8-dimercaptooctanoic and this is in accordance acid, with is a the strong observation antioxidant that lipoic due to acid hydrogen can scavenge transfer only [17 ,18]. very reactive radicals, while the reduced form, 6,8-dimercaptooctanoic acid, is a strong antioxidant Thus, it could be suggested that the reduced form of 6 may contribute to the in vivo anti-inflammatory due to hydrogen transfer [17,18]. Thus, it could be suggested that the reduced form of 6 may and hypolipidemic activity of this compound. contribute to the in vivo anti-inflammatory and hypolipidemic activity of this compound. Inhibition of Lipoxygenase Inhibition of Lipoxygenase Lipoxygenases,Lipoxygenases, involved involved in inflammation, in inflammation, are a are family a family of enzymes of enzymes that catalyse that the catalyse dioxygenation the of polyunsaturateddioxygenation of fatty polyunsaturated acids which fatty contain acids the whichcis-1,4-pentadiene contain the cis structure.-1,4-pentadiene Although structure. there are severalAlthough enzymes, there they are several all catalyse enzymes, the stereo-they all and catalyse regio-specific the stereo- peroxidation and regio-specific of arachidonic peroxidation or linoleicof acidarachidonic in the presence or linoleic of molecular acid in oxygen.the presence Soybean of molecular lipoxygenase-1 oxygen. canSoybean use arachidonic lipoxygenase-1 acid can as substrate,use witharachidonic about 15% acid of activity as substrate, for linoleic with acid.about It 15% has beenof activity found for that linoleic arachidonic acid. It acidhas bindingbeen found sites that in plant lipoxygenasesarachidonic share acid binding almost thesites same in plant similarity lipoxygenases with animal share 5-lipoxygenasealmost the same [similarity19]. 5-Lipoxygenase with animal activity5- contributes to atherosclerosis via oxidation of low-density lipoprotein. Furthermore, studies using MoleculesMolecules2019 2019, 24,, 24 3277, x 7 of6 12 of 12

lipoxygenase [19]. 5-Lipoxygenase activity contributes to atherosclerosis via oxidation of low-density 5-lipoxygenase-deficientlipoprotein. Furthermore, mice studies show that using 5-lipoxygenase 5-lipoxygenase-deficient activity may micecontribute show to that stress 5-lipoxygenase and depression behaviouractivity may [20]. contribute to stress and depression behaviour [20]. TheThe eff ect effect of of the the synthesised synthesised compounds compounds on on soybean lipoxygenase-1, lipoxygenase-1, using using linoleic linoleic acid acid as as substrate,substrate, is demonstratedis demonstrated in in Table Table4. In4. thisIn this table, table, the the inhibition inhibition o ff offeredered by by nordihydroguaiaretic nordihydroguaiaretic acid (NDGA),acid (NDGA), a potent a lipoxygenase potent lipoxygenase inhibitor, inhibitor, is included. is included. BHA and BHA trolox and could trolox not inhibit could not lipoxygenase inhibit evenlipoxygenase at concentrations even at much concentrations higher than much 300 µ M. higher In addition, than 300 no µΜ. inhibition In addition, was observed no inhibition when linoleic was observed when linoleic acid was used at 1 mM, a concentration higher than the saturating substrate acid was used at 1 mM, a concentration higher than the saturating substrate concentration, under concentration, under the same experimental conditions. The decline of inhibition by increasing the the same experimental conditions. The decline of inhibition by increasing the concentration of the concentration of the substrate indicates a competitive inhibition of lipoxygenase. substrate indicates a competitive inhibition of lipoxygenase. Table 4. Effect of compounds 1–9, BHT, ibuprofen, ketoprofen, tolfenamic acid and NDGA on Tablelipoxygenase 4. Effect a. of compounds 1–9, BHT, ibuprofen, ketoprofen, tolfenamic acid and NDGA on lipoxygenase a. IC50 (µΜ) or Compound IC (µM) or % Compound % Inhibition/µΜ50 µ 1 Inhibition14%/50 µΜ/ M 2 1 14%-/ 50 µM 3 2 84- 4 3 8684 4 86 5 22%/100 µΜ 5 22%/100 µM 6 6 6060 7 7 - - 8 8 4444 9 9 255255 BHTBHT 192 192 IbuprofenIbuprofen 200 200 KetoprofenKetoprofen 220 220 Tolfenamic acid 170 Tolfenamic acid 170 NDGA 1.3 NDGA 1.3 a After 7 min of incubation; BHT: 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene); NDGA: a nordihydroguaiaretic After 7 min of acid; incubation; -: inactive. BHT: 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene); NDGA: nordihydroguaiaretic acid; -: inactive.

TheThe time time course course of of lipoxygenase lipoxygenase inhibition by by the the most most active active of ofthe the synthesised synthesised compounds, compounds, 3 3 andand8 ,8, is is shown shown inin FigureFigure 33..

1.0 Compound 3 control IC50 = 84 μΜ 0.8 50μΜ

0.6 60μΜ Α (234nm) 0.4 75μΜ

100μΜ 0.2

0.0 0 60 120 180 240 300 360 420 Τime (s)

Figure 3. Cont.

MoleculesMolecules2019 2019, 24,, 327724, x 8 of7 12 of 12

1.2 Compound 8 control IC50 = 44 μΜ 1.0 10 mM 0.8 25 μΜ

Α (234nm) 0.6 50 μΜ 0.4 75 μΜ 0.2

0.0 0 60 120 180 240 300 360 420 Time (s)

FigureFigure 3. Time 3. Time course course of of lipoxygenase lipoxygenase inhibition inhibition byby variousvarious concentrations of of compounds compounds 3 and3 and 8. 8.

FromFrom the the presented presented results results it it could could be be observed observed that,that, when lorazepam lorazepam was was esterified esteriﬁed with with rigid rigid acids,acids, e.g., e.g., naproxen naproxen (2 ), (2), trolox trolox (7 (),7), indole-3-acetic indole-3-acetic acid acid (9), inhibition inhibition was was insignificant insigniﬁcant or orabsent, absent, whereas,whereas, less less rigid rigid substitution substitution seems seems to to contribute contribute toto strongerstronger inhibition, e.g., e.g., ketoprofen ketoprofen (3), (3 lipoic), lipoic acidacid (6), ( BHA6), BHA (8). (8).

3. Materials3. Materials and and Methods Methods

3.1.3.1. General General AllAll commercially commercially available available reagents reagents were were purchased purchased from from Merck Merck (Kenilworth, (Kenilworth, NJ, NJ, USA) USA) and and used withoutused further without purification. further purification.κ-Carrageenan κ-Carrageenan and lipoxygenase and lipoxygenase type I from type soybean I from weresoybean purchased were frompurchased Sigma (St. from Louis, Sigma MO, (St. USA). Louis, The MO, IR USA). spectra The were IR spectra recorded were on recorded a Perkin on Elmer a Perkin Spectrum Elmer BX FT-IRSpectrum spectrometer BX FT-IR (Waltham, spectrometer MA, (Waltham, USA). The MA,1H-NMR USA). spectraThe 1H-NMR were spectra recorded were using recorded an AGILENT using DD2-500an AGILENT MHz spectrometer DD2-500 MHz (Santa spectrometer Clara, CA, (Santa USA). Clara, All CA, chemical USA). All shifts chemical are reported shifts are in reportedδ (ppm) in and signalsδ (ppm) are givenand signals as follows: are given s, singlet; as follows: d, doublet; s, singlet; t, d, triplet; doublet; m, multiplet.t, triplet; m, Melting multiplet. points Melting (m.p.) points were (m.p.) were determined with a MEL-TEMPII Laboratory Devices, Sigma-Aldrich (Milwaukee WI, determined with a MEL-TEMPII Laboratory Devices, Sigma-Aldrich (Milwaukee WI, USA) apparatus USA) apparatus and are uncorrected. The microanalyses were performed on a Perkin-Elmer 2400 and are uncorrected. The microanalyses were performed on a Perkin-Elmer 2400 CHN elemental CHN elemental analyser. Wistar rats (160–220 g, 3–4 months old) were kept in the Centre of the analyser. Wistar rats (160–220 g, 3–4 months old) were kept in the Centre of the School of Veterinary School of Veterinary Medicine (EL54 BIO42), Aristotelian University of Thessaloniki, which is Medicine (EL54 BIO42), Aristotelian University of Thessaloniki, which is registered by the official state registered by the official state veterinary authorities (presidential degree 56/2013, in harmonization veterinarywith the authoritiesEuropean Directive (presidential 2010/63/EEC). degree 56The/2013, experimental in harmonization protocols were with approved the European by the Animal Directive 2010Ethics/63/EEC). Committee The experimental of the Prefecture protocols of Central were Macedonia approved (no. by 270079/2500). the Animal Ethics Committee of the Prefecture of Central Macedonia (no. 270079/2500). 3.2. Synthesis 3.2. Synthesis 3.2.1. General Method for the Synthesis of Compounds 1–9. General Method for the Synthesis of Compounds 1–9 A) In a solution of the corresponding acid (1mmol) in dichloromethane (CH2Cl2) lorazepam is suspendedA) In a solution (1.05 mmol) of the corresponding and N,N′-dicyclohexylcarbodiimide acid (1mmol) in dichloromethane (DCC, 1.3mmol) (CH was2Cl2 ) added. lorazepam The is suspendedreaction (1.05mixture mmol) was andstirredN,N for0-dicyclohexylcarbodiimide 4–12h. After filtration, the final (DCC, compounds 1.3mmol) were was isolated added. Thewith reaction flash mixturechromatography was stirred using for petroleum 4–12h. After ether filtration,and ethyl acetate the final as eluents. compounds were isolated with flash chromatographyB) For compound using petroleum 7: Troloxether (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic and ethyl acetate as eluents. acid, 1mmol) wasB) Fordissolved compound in tetrahydrofuran7: Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic (THF) and carbonyldiimidazole (CDI, 1.2 mmol) was acid, added. 1mmol) After was dissolvedstirring in for tetrahydrofuran 45 min, lorazepam (THF) (1.05 and mmol) carbonyldiimidazole was added and (CDI, the reaction 1.2 mmol) was was left added. for 12 Afterh at room stirring for 45temperature. min, lorazepam The solvent (1.05 was mmol) distilled was addedoff and and the residue the reaction was dissolved was left forin ethyl 12 h acetate at room and temperature. washed The solvent was distilled off and the residue was dissolved in ethyl acetate and washed with water. Molecules 2019, 24, 3277 8 of 12

The solution was dried over Na2SO4 and the ﬁnal compound was isolated with ﬂash chromatography using petroleum ether and ethyl acetate as eluents. 7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 2-(4-isobutylphenyl) propanoate (1). Flash chromatography (petroleum ether/ethyl acetate, 4/1). White solid, yield 74%, m.p. 105–122 ◦C. IR (nujol): 3297 (N-H), 1738, 1714 (C=O ester diastereomers), 1621 (C=O amide), 1527 (C-C 1 1 aromatic) cm− . H-NMR (CDCl3) δ: 0.92 (d, 6H, J = 6.4 Hz, CH3-CH-CH3), 1.65 (dd, 3H, J = 14.1, 7.1 Hz, -CO-CH-CH3), 1.95–1.78 (m, 1H, CH3-CH-CH3), 2.46 (d, 2H, J = 7.1 Hz, -CH2CH-(CH3)2), 4.05 (q, 1H, J = 7.1 Hz, -CO-CH-CH3), 6.02, 6.00 (s, 1H, -O-CH-C=O), 7.17–7.00 (m, 4H, aromatic ibuprofen), 7.65–7.30 (m, 7H, aromatic lorazepam), 9.21 (s, 1H, -NH). Anal. Calcd for C28H26Cl2N2O3: C, 66.02; H, 5.14; N, 5.50. Found: C, 65.82; H, 5.30; N, 5.33. 7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 2-(6-methoxynaphthalen-2-yl)propanoate (2). Flash chromatography (petroleum ether/ethyl acetate, 3/1). White solid, yield 93%, m.p. 136–139 ◦C. IR (nujol): 3220 (N-H), 1702 (C=O ester), 1 1 1627 (C=O amide), 1605, 1569 (C-C aromatic) cm− . H-NMR (CDCl3) δ: 1.75 (d, 3H, J = 7.7 Hz, CH3-CH-C=O), 3.94 (s, 3H, -O-CH3), 4.28–4.10 (m, 1H, CH3-CH-C=O), 6.01 (s, 1H, -O-CH-C=O), 6.99 (d, 1H, J = 8.5 Hz, naphthyl C2), 7.04 (s, 1H, naphthyl C10), 7.18–7.08 (m, 2H, naphthyl C7, C8), 7.45–7.25 (m, 3H, chlorophenyl C3, C4, C5 and naphthyl C5), 7.52 (d, 1H, J = 8.5 Hz, naphthyl C3), 7.76–7.55 (m, 3H, chlorophenyl C6 and chlorophenylamino C3, C5), 7.83 (d, 1H, J = 8.1 Hz, chlorophenylamino C6), 9.42 (s, 1H, O=C-NH-). Anal. Calcd for C29H22Cl2N2O4: C, 65.30; H, 4.16; N, 5.25. Found: C, 65.42; H, 4.26; N, 4.89. 7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 2-(3-benzoylphenyl)propanoate (3). Flash chromatography (petroleum ether/ethyl acetate, 3/1).White solid, yield 85%, m.p.195–201 ◦C. IR (nujol): 3203 (N-H), 1739 (C=O ketone), 1712, 1696 (C=O 1 1 ester diastereomers), 1650 (C=O amide), 1596 (C-C aromatic) cm− . H-NMR (CDCl3 + DMSO-d6) δ: 1.73–1.67 (m, 3H, -CO-CH-CH3), 4.20–4.08 (m, 1H, -CO-CH-CH3), 6.04, 6.02 (s, 1H, -O-CH-C=O), 7.15–7.00 (m, 2H, isopropylphenyl C5, chlorophenyl C5), 7.93–7.33 (m, 14H, aromatic), 8.70, 8.73 (s, 1H, NH-). Anal. Calcd for C31H22Cl2N2O4: C, 66.80; H, 3.98; N, 5.03. Found: C, 66.54; H, 3.91; N, 4.67. 7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 2-(1-(4-chlorobenzoyl)- 5-methoxy-2-methyl-1H-indol-3-yl)acetate (4). Flash chromatography (petroleum ether/ethyl acetate, 2/1). White solid, yield 81%, m.p. 189–191 ◦C. IR (nujol): 3302 (N-H), 1748 (C=O ester), 1699 (C=O 1 1 amide indole), 1620 (C=O amide lorazepam), 1574 (C-C aromatic) cm− . H-NMR (CDCl3 + DMSO-d6) δ: 2.25 (s, 3H, CH3-C-N-), 3.71 (s, 3H, -O-CH3), 3.83 (s, 2H, -CH2-C=O), 5.84 (s, 1H, -O-CH-C=O), 6.55 (d, 1H, J = 9.0 Hz, indole C6), 6.85 (d, 1H, J = 9.0 Hz, indole C7), 6.89 (s, 1H, indole C4), 6.98 (s, 1H, chlorophenylamino C3), 7.13 (d, 1H, J = 8.7 Hz, chlorophenyl C3), 7.31–7.21 (m, 4H, chlorophenylamino C5 and chlorophenyl C4, C5, C6), 7.33 (d, 2H, J = 8.4 Hz, aromatic chloro-benzoyl C3, C5), 7.41 (d, 1H, J = 6.0 Hz, chlorophenylamino C6), 7.54 (d, 2H, J = 8.4 Hz, chlorobenzoyl C2, C6), 10.61 (s, 1H, -NH-). Anal. Calcd for C34H24Cl3N3O5: C, 61.79; H, 4.56; N, 6.36. Found: C, 61.65; H, 4.22; N, 5.92. 7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 2-((3-chloro-2-methylphenyl)amino)benzoate (5). Flash chromatography (petroleum ether/ethyl acetate, 2/1). White solid, yield 41%, m.p. 206–207 ◦C. IR (nujol): 3327 (N-H), 1721 (C=O ester), 1635 1 1 (C=O amide), 1583 (C-C aromatic) cm− . H-NMR (CDCl3 + DMSO-d6) δ: 2.25 (s, 3H, -CH3), 6.14 (s, 1H, -O-CH-C=O), 6.80–6.67 (m, 2H, aromatic amino methyl phenyl C6 and aromatic benzoic acid C5), 7.45–6.96 (m, 10H, aromatic chloro-methyl-phenylamino C4, C5, benzoic acid C3, C4, C6, chlorophenylamino C3, C5 and chlorophenyl C3, C4, C5), 7.55 (d, 1H, J = 7.6 Hz, chlorophenyl C6), 8.25 (d, 1H, J = 8.1 Hz, chlorophenylamino C6), 9.09 (s, 1H, -NH-), 10.93 (s, 1H, O=C-NH-). Anal. Calcd for C29H20Cl3N3O3: C, 61.66; H, 3.57; N, 7.44. Found: C, 62.03; H, 3.53; N, 7.81. Molecules 2019, 24, 3277 9 of 12

7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 5-(1,2-dithiolan -3-yl)pentanoate (6). Flash chromatography (petroleum ether/ethyl acetate, 1/1). White solid, yield 71%, m.p. 184–189 ◦C. IR (nujol): 3237 (N-H), 1713, 1682 (C=O ester), 1620 (C=O amide), 1592 (C-C 1 1 aromatic) cm− . H-NMR (CDCl3 + DMSO-d6) δ: 1.80–1.67 (m, 2H, C3 H), 1.62–1.47 (m, 4H, C4, C5), 1.93–1.84 (m, 1H, C7 axial), 2.33–2.21 (m, 1H, C7 equatorial), 2.43–2.35 (m, 2H, C2), 3.03–2.86 (m, 2H, C6, C8 axial), 3.42–3.32 (m, 1H, C8 equatorial), 5.75 (s, 1H, -O-CH-C=O), 6.83 (d, 1H, J = 2.3 Hz, chlorophenyl C3), 7.09 (d, 1H, J = 8.7 Hz, chlorophenyl C6), 7.28–7.17 (m, 4H, chlorophenylamino C3, C5 and chlorophenyl C4, C5), 7.39–7.34 (m, 1H, chlorophenylamino C6), 10.64 (s, 1H, O=C-NH-). Anal. Calcd for C23H22Cl2N2O3S2: C, 54.22; H, 4.35; N, 5.50. Found: C, 54.41; H, 4.68; N, 5.33. 7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylate (7). Flash chromatography (petroleum ether/ethyl acetate, 3/2). White solid, yield 85%, m.p. 138–144 ◦C. IR (nujol): 3400 (O-H), 3250 (N-H), 1680 (C=O ester), 1631 1 1 (C=O amide) cm− . H-NMR (CDCl3) δ: 1.78 (s, 3H, 2-CH3), 2.00 (s, 3H, 5-CH3), 2.10 (s, 3H, 7-CH3), 2,15 (s, 3H, 8–CH3), 2.20–2.65 (m, 4H, chromane), 5.10 (s, 1H, -OH), 6.10 (s, 1H, -O-CH-C=O), 7.20 - 7.82 (m, 7H, aromatic), 9.40 (s, 1H, -NH-). Anal. Calcd for C29H26Cl2N2O5: C, 62.94; H, 4.74; N, 5.06. Found: C, 62.69; H, 4.96; N, 5.13. (E)-7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 3-(3,5-di-tert-butyl- 4-hydroxyphenyl)acrylate (8). Flash chromatography (petroleum ether/ethyl acetate, 6/1 and then 3/1). White solid, yield 40%, m.p. 274 ◦C. IR (nujol): 3611 (O-H), 3277 (N-H), 1710 (C=O ester), 1627 (C=O 1 1 amide), 1593 (C-C aromatic) cm− . H-NMR (CDCl3 + DMSO-d6) δ: 1.49 (s, 18H, -CH3), 5.56 (s, 1H, phenol OH), 6.00 (s, 1H, -O-CH-C=O), 6.61 (d, 1H, J = 15.9 Hz, CH=CH-C=O), 7.89 (d, 1H, J = 15.9 Hz, CH=CH-C=O), 7.13 (d, 1H, J = 2.2 Hz, chlorophenyl C6), 7.21 (d, 1H, J = 8.7 Hz, chlorophenyl C3), 7.51–7.39 (m, 6H, chlorophenyl C4, C5, chlorophenylamino C3, C5 and di-tert-butylphenyl C2, C6), 7.64 (d, 1H, J = 7.4 Hz, chlorophenylamino C6), 7.89 (d, 1H, J = 15.9 Hz, CH=CH-C=O), 8.97 (s, 1H, -NH-). Anal. Calcd for C32H32Cl2N2O4 x0.7CH2Cl2: C, 61.47; H, 5.27; N, 4.38. Found: C, 61.31; H, 5.58; N, 4.06. 7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 2-(1H-indol-3-yl)acetate (9). Flash chromatography (petroleum ether/ethyl acetate, 2/1). White solid, yield 40%, m.p. 257–258 ◦C. 1 1 IR (nujol): 3360 (N-H), 1760 (C=O ester), 1713 (C=O amide), 1621 (C-C aromatic) cm− . H-NMR (CDCl3 + DMSO-d6) δ: 3.96 (s, 1H, -CH2-C=O), 5.89 (s, 1H, -O-CH-C=O), 6.94 (d, 1H, J = 2.2 Hz, indole C7), 6.99 (d, 1H, J = 7.5 Hz, indole C6), 7.06 (t, 1H, J = 7.5 Hz, indole C6), 7.25–7.17 (m, 2H, aromatic indole C2, C4), 7.59–7.28 (m, 7H, aromatic lorazepam), 10.16 (s, 1H, indole -NH-), 10.89 (s, 1H, O=C-NH-). Anal. Calcd for C25H17Cl2N3O3 x2.4H2O: C, 57.57; H, 4.21; N, 8.06. Found: C, 57.53; H, 3.88; N, 8.12.

3.3. Eﬀect on Carrageenan-Induced Rat Paw Oedema An aqueous solution of carrageenan was prepared (1% w/v) and 0.1 mL of this was injected i.d. into the right hind paw of female rats; the left paw served as control. The tested compounds (suspended in water with a few drops of Tween 80) were given intra peritoneally (i.p.) (0.15 mmol/kg) 5 min prior to the carrageenan injection. After 3.5 h the hind paws were weighed separately. The produced oedema was estimated as paw weight increase [18].

3.4. Eﬀect on Plasma Total Cholesterol, Triglyceride and LDL-cholesterol Levels A solution of Triton WR 1339 (tyloxapol) in saline was administered i.p. (200 mg/kg) to male rats and 1 h later the examined compound (0.15 and/or 0.05 mmol/kg, suspended in water with a few drops of Tween 80) was given i.p. After 24 h, blood was taken from the aorta and used for the determination of plasma total cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) concentrations, using commercial kits, against standard solutions [21]. Molecules 2019, 24, 3277 10 of 12

3.5. Eﬀect on Lipid Peroxidation The incubation mixture contained heat-inactivated rat hepatic microsomal fraction, ascorbic acid (0.2 mM) in Tris–HCl/KCl buﬀer (pH 7.4) and the test compounds dissolved in dimethylsulphoxide. The reaction was initiated by FeSO4 (10 µM) and the mixture was incubated at 37 ◦C. Aliquots were taken at various time intervals for 45 min. Lipid peroxidation was assessed spectrophotometrically (535 against 600 nm) by the determination of 2-thiobarbituric acid (TBA) reactive material [22].

3.6. Interaction with the Stable Radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) Compounds (in absolute ethanol, ﬁnal concentration 50–200 µM) were added to an equal volume of an ethanolic solution of DPPH (ﬁnal concentration 200 µM) at room temperature (22 2 C). ± ◦ Absorbance (517 nm) was recorded after 30 min [22].

3.7. Eﬀect on Lipoxygenase Activity The reaction mixture contained the test compounds dissolved in ethanol and soybean lipoxygenase (250 U/mL) in Tris buﬀer (pH 9). The reaction was initiated by the addition of sodium linoleate (0.1 mM) and monitored for 7 min at 28 ◦C, by recording the absorbance of a conjugated diene structure at 234 nm. For the estimation of the type of inhibition, the above experiments were repeated, using sodium linoleate at 1 mM, which is higher than the saturating substrate concentration [18].

4. Conclusions In conclusion, the synthesised compounds presented important hypolipidemic activity and significant in vivo anti-inflammatory properties, being superior to the parent acids. It is reported that anxiolytic therapy improves stress-induced inflammation [23]. Furthermore, inflammation, endothelial dysfunction and platelet aggregation may connect anxiety disorders with cardiovascular diseases [24]. Finally, we have shown that molecules containing lorazepam and antioxidants, linked by a GABA residue, reduced stress, caused by immobilization and fasting, as well as the subsequent oxidative damage [7]. Complex stress-related disorders could be treated effectively with agents designed to act at different stages of their pathogenesis. The present research may provide useful candidate molecules towards this target.

Author Contributions: Investigation, P.T.-N. and G.P.; Supervision, E.A.R.; Writing—review & editing, P.N.K. Funding: This research received no external funding Acknowledgments: G.P. acknowledges the General Secretariat for Research and Technology (GSRT) of Greece and the Hellenic Foundation for Research and Innovation (HFRI) for a grant supporting his PhD research. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds 1–9 are available from the authors.

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